The temporal regulation of replication origin initiation timing is a form of epigenetic regulation whose significance and mechanism remain poorly understood. The origin timing program is thought to be connected with the gene expression program during development, perhaps to ensure the stable inheritance of transcriptional states. Differential origin timing is also propose to play a role in genome stability. Chromatin structure has been implicated in modulating replication timing but a precise mechanism remains vague. We have now determined that Forkhead transcription factors, Fkh1 and Fkh2 (Fkh1/2), play a major role in establishing genomic replication patterns by determining the activation of many of the most efficient, early-firing origins in the yeast genome. No other factor has been identified as having such a significant impact, in level and breadth, on genomic replication patterns. The role of Fkh1/2 in origin regulation is independent of their roles in transcriptional regulation. Instead, our results show that Fkh1/2 are required for early origin clustering or subnuclear localization and their association with the initiation factor Cdc45, both in G1-phase. These results suggest the overall hypothesis that Forkhead proteins play a key role in origin regulation by tethering origins into clusters that will become the first replication factories in S-phase. We propose that origin clustering is mediated by a novel mechanism of Fkh1/2 dimerization and/or by interactions with ORC, which we recently demonstrated. These findings have opened exciting new avenues toward understanding genome organization and mechanisms of epigenetic regulation. This proposal will examine new mechanisms of function for Forkhead transcription factors in replication that will inform our understanding of their roles in transcription regulation, and our newly hypothesized role in higher-order genome organization. Our Specific Aims to determine the mechanisms that Fkh1 and Fkh2 use to regulate chromosome replication and structure are: 1) Develop molecular tools to analyze and manipulate origin clustering dynamics 2) Elucidate the role of Fkh1/2 binding in the mechanism of origin regulation 3) Perform structure-function analysis of Fkh1 and Fkh2 4) Characterize the long-range chromatin interactions mediated by Fkh1 and Fkh2